Humanization of Yeast to Produce Complex Terminally Sialylated Glycoproteins

Hamilton, Stephen R.; Davidson, Robert C.; Sethuraman, Natarajan; Nett, Juergen H.; Jiang, Yi; Rios, Sandra; Bobrowicz, Piotr; Stadheim, Terrance A.; Li, Huijuan; Choi, Byungcho; Hopkins, Daniel; Wischnewski, Harry; Roser, Jessica; Mitchell, Teresa; Strawbridge, Rendall R.; Hoopes, Jack; Wildt, Stefan; Gerngross, Tillman U.

doi:10.1126/science.1130256

Cited by 463 publications

(283 citation statements)

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“…3b) [4][5][6] . The heterologous glycosyltransferases needed for this use the sugar-nucleotides UDP-GlcNAc and UDP-Gal as monosaccharide donors.…”

Section: Discussionmentioning

confidence: 99%

“…Several P. pastoris-produced biopharmaceuticals that are either not glycosylated (such as human serum albumin 2 ) or for which glycosylation is needed only for proper folding (such as several vaccines 3 ) are already on the market. An important recent breakthrough has been the development of P. pastoris strains with humantype N-glycosylation [4][5][6] . Humanized glycosylation will further increase the importance of P. pastoris for biopharmaceutical production; indeed, proteins produced with this system are moving into clinical development 7 .…”

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confidence: 99%

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Genome sequence of the recombinant protein production host Pichia pastoris

et al. 2009

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The methylotrophic yeast Pichia pastoris is widely used for the production of proteins and as a model organism for studying peroxisomal biogenesis and methanol assimilation. P. pastoris strains capable of human-type N-glycosylation are now available, which increases the utility of this organism for biopharmaceutical production. Despite its biotechnological importance, relatively few genetic tools or engineered strains have been generated for P. pastoris. To facilitate progress in these areas, we present the 9.43 Mbp genomic sequence of the GS115 strain of P. pastoris. We also provide manually curated annotation for its 5,313 protein-coding genes.The methylotrophic yeast Pichia pastoris is by far the most commonly used yeast species in the production of recombinant proteins 1 and is employed in laboratories around the world to produce proteins for basic research and medical applications. It is also an important model organism for the investigation of peroxisomal proliferation and methanol assimilation. The P. pastoris expression technology has been commercially available for many years. P. pastoris grows to high cell density, provides tightly controlled methanol-inducible transgene expression and efficiently secretes heterologous proteins in defined media. Several P. pastoris-produced biopharmaceuticals that are either not glycosylated (such as human serum albumin 2 ) or for which glycosylation is needed only for proper folding (such as several vaccines 3 ) are already on the market. An important recent breakthrough has been the development of P. pastoris strains with humantype N-glycosylation [4][5][6] . Humanized glycosylation will further increase the importance of P. pastoris for biopharmaceutical production; indeed, proteins produced with this system are moving into clinical development 7 . Moreover, monoclonal antibodies can be made at gramper-liter scale in the humanized glycosylation-homogenous strains 8 .For further strain engineering, a better understanding of all aspects of the yeast's protein production machinery is needed, and a number of studies relating to P. pastoris's secretory system and engineered promoters have been forthcoming 9,10 . To facilitate the investigation of P. pastoris and other methylotrophic yeasts, we present the 9.43 Mbp genomic sequence of the GS115 strain of P. pastoris.

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“…3b) [4][5][6] . The heterologous glycosyltransferases needed for this use the sugar-nucleotides UDP-GlcNAc and UDP-Gal as monosaccharide donors.…”

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Genome sequence of the recombinant protein production host Pichia pastoris

et al. 2009

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“…The application of a combinatorial library approach has been essential to generate P. pastoris strains harboring combinations of mannosidases, glycosyltransferases, GlcNAc/Gal transporters, and Gal epimerase [24]. Over the past years we have created a library of glycoengineered P. pastoris strains each capable of displaying defined N-linked glycans at high uniformity [5,8,12,17,24]. This growing library of strains has the potential to allow elucidation of the relationship between specific N-linked glycans and the function of glycoproteins.…”

Section: Introductionmentioning

confidence: 99%

Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response

et al. 2008

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Traditional production of therapeutic glycoproteins relies on mammalian cell culture technology. Glycoproteins produced by mammalian cells invariably display N-glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. However, extent and type of N-glycosylation has a profound impact on the therapeutic properties of many commercially relevant therapeutic proteins making control of N-glycosylation an emerging field of high importance. We have employed a combinatorial library approach to generate glycoengineered Pichia pastoris strains capable of displaying defined human-like N-linked glycans at high uniformity. The availability of these strains allows us to elucidate the relationship between specific N-linked glycans and the function of glycoproteins. The aim of this study was to utilize this novel technology platform and produce two human-like N-linked glycoforms of recombinant human lactoferrin (rhLF), sialylated and non-sialylated, and to evaluate the effects of terminal N-glycan structures on in vitro secondary humoral immune responses. Lactoferrin is considered an important first line defense protein involved in protection against various microbial infections. Here, it is established that glycoengineered P. pastoris strains are bioprocess compatible. Analytical protein and glycan data are presented to demonstrate the capability of glycoengineered P. pastoris to produce fully humanized, active and immunologically compatible rhLF. In addition, the biological activity of the rhLF glycoforms produced was tested in vitro revealing the importance of N-acetylneuraminic (sialic) acid as a terminal sugar in propagation of proper immune responses.

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“…Quel que soit le système considéré, plusieurs stratégies ont été déve-loppées pour corriger les différences observées de glycosylation entre protéines naturelles et recombinantes, soit par l'inactivation de gènes codant pour des enzymes introduisant les résidus immunogènes, soit par complémentation par des gènes codant des enzymes permettant de reconstruire les séquences glucidiques absentes dans ces organismes [5]. Les résultats les plus spectaculaires ont été obtenus par Gerngross et ses collaborateurs, au sein de la société GlycoFi (États-Unis), qui ont complètement « humanisé » la N-glycosylation chez la levure par la suppression de 4 gènes et l'ajout de 14 autres [6].…”

Section: Chloroplastesunclassified